{"title":"Development and validation of a UHPLC-MS/MS method for the quantitative analysis of trans-ISRIB in human plasma.","authors":"Weizhuan He, Akshay Suresh Patil, Yan Xu","doi":"10.1016/j.jchromb.2025.124469","DOIUrl":null,"url":null,"abstract":"<p><p>The integrated stress response (ISR) is a cellular defense mechanism activated under stress conditions. When the ISR is activated, it slows the production of proteins, the building blocks that cells need to function. Trans-integrated stress response inhibitor (trans-ISRIB) is a compound that can reverse the effects of ISR activation, showing promise for treating neurodegenerative diseases. The preclinical and clinical evaluation of trans-ISRIB necessitates a reliable analytical method. This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, conforming to the U.S. FDA's guidelines for bioanalytical method validation. The method developed utilizes a liquid-liquid extraction procedure to prepare plasma samples with a spiked internal standard (IS). The extracts containing trans-ISRIB and the IS were dried under nitrogen, reconstituted in the mobile phase, and separated on a Waters XSelect HSS T3 column under isocratic conditions with a mobile phase containing 0.1 % acetic acid in 70 % methanol aqueous solution at a flow rate of 0.500 mL/min. Detection and quantification were accomplished using a positive electrospray ionization tandem mass spectrometer (ESI<sup>+</sup>-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The method demonstrated a linear calibration range for trans-ISRIB concentrations from 0.500 to 1.00 x 10<sup>3</sup> nM, with high specificity, precision, accuracy, and recovery. This method addresses a significant analytical gap, offering a robust tool for quantifying trans-ISRIB in human plasma. Chemical compounds studied in this article: 2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide (trans-ISRIB) (CAS # 1597403-47-8); 2-(4-chlorophenoxy)-N-(2-{[(4-chlorophenoxy)acetyl]amino}ethyl)acetamide (CAS # 327071-30-7).</p>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"124469"},"PeriodicalIF":2.8000,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Chromatography B","FirstCategoryId":"1","ListUrlMain":"https://doi.org/10.1016/j.jchromb.2025.124469","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
The integrated stress response (ISR) is a cellular defense mechanism activated under stress conditions. When the ISR is activated, it slows the production of proteins, the building blocks that cells need to function. Trans-integrated stress response inhibitor (trans-ISRIB) is a compound that can reverse the effects of ISR activation, showing promise for treating neurodegenerative diseases. The preclinical and clinical evaluation of trans-ISRIB necessitates a reliable analytical method. This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, conforming to the U.S. FDA's guidelines for bioanalytical method validation. The method developed utilizes a liquid-liquid extraction procedure to prepare plasma samples with a spiked internal standard (IS). The extracts containing trans-ISRIB and the IS were dried under nitrogen, reconstituted in the mobile phase, and separated on a Waters XSelect HSS T3 column under isocratic conditions with a mobile phase containing 0.1 % acetic acid in 70 % methanol aqueous solution at a flow rate of 0.500 mL/min. Detection and quantification were accomplished using a positive electrospray ionization tandem mass spectrometer (ESI+-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The method demonstrated a linear calibration range for trans-ISRIB concentrations from 0.500 to 1.00 x 103 nM, with high specificity, precision, accuracy, and recovery. This method addresses a significant analytical gap, offering a robust tool for quantifying trans-ISRIB in human plasma. Chemical compounds studied in this article: 2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide (trans-ISRIB) (CAS # 1597403-47-8); 2-(4-chlorophenoxy)-N-(2-{[(4-chlorophenoxy)acetyl]amino}ethyl)acetamide (CAS # 327071-30-7).
期刊介绍：
The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis.
Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches.
Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.
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